Stimulating autophagy — a major cellular clearance route for intracellular protein aggregates — could represent a therapeutic strategy for Huntington's disease. However, the only drug that has been shown to induce autophagy in the brain is rapamycin, a mammalian target of rapamycin (mTOR) kinase inhibitor that modulates several cellular processes besides autophagy. Now, Williams and colleagues have identified a new mTOR-independent autophagy pathway that provides novel targets for Huntington's disease.
First, using cell-based assays, the authors screened a library of 253 compounds that included FDA-approved drugs to identify autophagy enhancers. Hits included minoxidil (an ATP-sensitive K+ channel agonist); clonidine (an α2-adrenergic and type I imidazoline receptor agonist); and verapamil (an L-type Ca2+ channel antagonist). These compounds enhanced clearance of soluble mutant huntingtin and reduced its aggregation and toxicity in neuronal cells in an autophagy-dependent manner.
Next, the authors identified calpains — Ca2+-dependent proteases that are enhanced in Huntington's disease — as central players in autophagy regulation. These proteases were common targets of the two major pathways identified in the primary screen: the established cAMP–Epac–PLC–IP3 pathway (inhibited by clonidine signalling via the imidazoline receptor) and the L-type Ca2+ channel pathway. A calpain inhibitor was shown to reduce huntingtin aggregation and increase autophagic activity in transfected cells. In addition, calpains were found to act downstream of L-type Ca2+ channels in this pathway.
To identify the substrates for calpains that regulate autophagy, the authors studied the α-subunit of the heterotrimeric G protein Gs, which is cleaved by calpains resulting in constitutive activation. They showed that Gsα inhibition decreased huntingtin aggregation and increased autophagosome synthesis, and that calpain inhibitors had no effects in cells with Gsα knockdown. This suggested that Gsα is likely to mediate many of the autophagy-related effects of calpains, and creates a link between the cAMP–Epac–PLC–IP3 pathway and the Ca2+–calpain pathway.
Several lines of evidence suggested that calpain-regulated autophagy was mTOR independent: induction of autophagy by inhibiting calpains occurred even when mTOR is activated. In addition, calpain inhibitors and rapamycin had additive effects in reducing huntingtin aggregation and toxicity.
Compounds were then tested in Drosophila and zebrafish models of Huntington's disease. In Drosophila, verapamil and clonidine had protective effects, and zebrafish treated with these drugs had fewer huntingtin aggregates.
This study reveals a new cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating Gsα, which regulates cAMP levels. Importantly, this pathway has numerous potential points where autophagy can be induced, and so drugs acting on this pathway — including some already approved for clinical use — may have added efficacy in combination with rapamycin for neurodegenerative diseases.
ORIGINAL RESEARCH PAPER
Williams, A. et al. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nature Chem. Biol. 4, 295–305 (2008)
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Human Molecular Genetics (2012)
Japanese Journal of Thrombosis and Hemostasis (2008)